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Sterilisation and
Sterilisation and
Disinfection
Disinfection
Dr. Pendru Raghunath Reddy
Dr. Pendru Raghunath Reddy
Why we need Sterilization?
Why we need Sterilization?
 Microorganisms capable of causing infection are
Microorganisms capable of causing infection are
constantly present in the external environment and on
constantly present in the external environment and on
the human body
the human body
 Microorganisms are responsible for contamination and
Microorganisms are responsible for contamination and
infection
infection
 The aim of sterilisation is to remove or destroy them
The aim of sterilisation is to remove or destroy them
from materials or from surfaces
from materials or from surfaces
Definitions:
Definitions:
Sterilisation :
Sterilisation :
– It is a process by which an article, surface or
It is a process by which an article, surface or
medium is made free of all microorganisms either
medium is made free of all microorganisms either
in vegetative or spore form
in vegetative or spore form
Disinfection :
Disinfection :
– Destruction of all pathogens or organisms capable
Destruction of all pathogens or organisms capable
of producing infections but not necessarily spores.
of producing infections but not necessarily spores.
– All organisms may not be killed but the number is
All organisms may not be killed but the number is
reduced to a level that is no longer harmful to
reduced to a level that is no longer harmful to
health.
health.
Antiseptics :
Antiseptics :
 Chemical disinfectants which can safely applied to
Chemical disinfectants which can safely applied to
living tissues and are used to prevent infection by
living tissues and are used to prevent infection by
inhibiting the growth of microorganisms
inhibiting the growth of microorganisms
Asepsis :
Asepsis :
 Technique by which the occurrence of infection into
Technique by which the occurrence of infection into
an uninfected tissue is prevented.
an uninfected tissue is prevented.
Methods
Methods
1.
1. Physical
Physical
methods
methods
2.
2. Chemical
Chemical
methods
methods
5
Physical methods:
Physical methods:
 Physical
Physical
methods:
methods:
1.
1. Sunlight
Sunlight
2.
2. Drying
Drying
3.
3. Heat
Heat
1.
1. Dry heat
Dry heat
2.
2. Moist heat
Moist heat
4.
4. Filtration
Filtration
5.
5. Radiation
Radiation 6
Chemical methods
Chemical methods
• Chemical methods:
Chemical methods:
1.
1. Alcohols
Alcohols
2.
2. Aldehydes
Aldehydes
3.
3. Phenols
Phenols
4.
4. Halogens
Halogens
5.
5. Oxidizing agents
Oxidizing agents
6.
6. Salts
Salts
7.
7. Surface active agents
Surface active agents
8.
8. Dyes
Dyes
9.
9. Vapor phase disinfectants
Vapor phase disinfectants
Physical methods
1. Sunlight
Sunlight possesses appreciable bactericidal activity
Due to its content of ultraviolet rays & heat rays
Under natural conditions, its sterilising power varies according to circumstances
Natural method of sterilisation of water in tanks, rivers and lakes
2. Drying
Moisture is essential for the growth of bacteria
Drying in air has a deleterious effect on many bacteria
This method is unreliable and is only of theoretical interest
3. Heat
Most reliable method of sterilisation
The factors influencing sterilisation by heat are:
Nature of heat
Temperature and time
Number of microorganisms present
Characteristics of the organisms, such as species, strain and sporing capacity
Type of material from which the organisms have to be eradicated
Mechanism of action
Dry heat
Kills organisms by protein denaturation, oxidative damage
and toxic effects of elevated levels of electrolytes
Moist heat
Kills microorganisms by coagulation and denaturation
of their enzymes and structural proteins
 Dry heat:
Dry heat:
1.
1. Red heat
Red heat
2.
2. Flaming
Flaming
3.
3. Incineration
Incineration
4.
4. Hot air oven
Hot air oven
Red heat
Red heat
Materials are held in
Materials are held in
the flame of a bunsen
the flame of a bunsen
burner till they become
burner till they become
red hot.
red hot.
 Inoculating wires or
Inoculating wires or
loops
loops
 Tips of forceps
Tips of forceps
 Surface of searing
Surface of searing
spatulae
spatulae
 Needles
Needles
Flaming
Flaming
Materials are passed
Materials are passed
through the flame of a
through the flame of a
bunsen burner without
bunsen burner without
allowing them to
allowing them to
become red hot.
become red hot.
 Glass slides
Glass slides
 scalpels
scalpels
 Mouths of culture
Mouths of culture
tubes and bottles
tubes and bottles
Incineration:
Incineration:
Materials are reduced
Materials are reduced
to ashes by burning.
to ashes by burning.
Instrument used was
Instrument used was
incinerator.
incinerator.
Soiled dressings
Soiled dressings
Animal carcasses
Animal carcasses
Bedding
Bedding
Pathological material
Pathological material
Hot air oven
Most widely used method of sterilisation by dry heat
It is used to process materials which can withstand high
temperatures, but which are likely to be affected by contact
with steam
It is a method of choice for sterilisation of glassware, forceps,
scissors, scalpels, swab sticks packed in test tubes
Materials such as oils, jellies and powders which are
impervious to steam are sterilised by hot air oven
Hot air oven is electrically heated and is fitted with a termostat
that maintains the chamber air at a chosen temperature
Fitted with a fan that distributes hot air in the chamber
Hot air oven
Holding temperature & time
General purpose
Temperature and time:
1600
C for 2 hours
1700
C for 1 hour
1800
C for 30 minutes
Cutting instruments such as those used in ophthalmic surgery,
Should be sterilized at 1500
C for 2 hours
Oils, glycerol and dusting powder should be sterilised at
1500
C for 1 hour
Precautions
Precautions
1.
1. Should not be overloaded
Should not be overloaded
2.
2. Arranged in a manner which allows free circulation of
Arranged in a manner which allows free circulation of
air
air
3.
3. Material to be sterilized should be perfectly dry.
Material to be sterilized should be perfectly dry.
4.
4. Test tubes, flasks etc. should be fitted with cotton
Test tubes, flasks etc. should be fitted with cotton
plugs.
plugs.
5.
5. petridishes and pipetts should be wrapped in paper.
petridishes and pipetts should be wrapped in paper.
6.
6. Rubber materials and inflammable materials should
Rubber materials and inflammable materials should
not be kept inside.
not be kept inside.
7.
7. The oven must be allowed to cool for two hours
The oven must be allowed to cool for two hours
before opening, since glass ware may crack by
before opening, since glass ware may crack by
sudden cooling.
sudden cooling.
Uses of Hot Air Oven
Uses of Hot Air Oven
 Sterilisation of
Sterilisation of
1.
1. Glassware like glass syringes, petri
Glassware like glass syringes, petri
dishes, pipettes and test tubes.
dishes, pipettes and test tubes.
2.
2. Surgical instruments like scalpels,
Surgical instruments like scalpels,
scissors, forceps etc.
scissors, forceps etc.
3.
3. Chemicals like liquid paraffin, fats etc.
Chemicals like liquid paraffin, fats etc.
Sterilisation controls
Sterilisation controls
1.
1. Spores of
Spores of Bacillus
Bacillus
subtilis subsp. Niger
subtilis subsp. Niger
2.
2. Thermocouples
Thermocouples
3.
3. Browne’s tube
Browne’s tube
(Tube containing red
(Tube containing red
colour solution is inserted
colour solution is inserted
in each load and a colour
in each load and a colour
change from red to green
change from red to green
indicates proper
indicates proper
sterilization)
sterilization)
Moist heat
Moist heat is divided into three forms
1.Temperature below 1000
C
2.At a temperature of 1000
C
3.Temperature above 1000
C
Temperatures below 1000
C
1. Pasteurization
2. Vaccine bath
3. Water bath
4. Inspissation
5. Low temperature steam-formaldehyde (LTSF) sterilization
Pasteurization
 Milk is sterilised by this method; Two methods
1.Holder method (630
C for 30 min followed by rapid
cooling to 130
C or lower)
2. Flash method (720
C for 15-20 seconds followed by rapid
cooling to 130
C or lower)
The dairy industry sometimes uses ultrahightemperature (UHT)
sterilization (140 to 1500
C for 1-3 seconds followed by rapid
cooling to 130
C or lower)
All nonsporing pathogens such as mycobacteria, brucellae
and salmonellae are destroyed by these processes
Coxiella burnetii is relatively heat resistant and may survive
the holder method
Principle of Pasteurization
Principle of Pasteurization
03/09/14 Dr.T.V.Rao MD
24
Vaccine bath
Vaccines prepared from nonsporing bacteria may be inactivated
in a water bath at 600
C for 1 hour
Water bath
Serum or body fluids containing coagulable proteins can be
sterilized by heating for 1 hour at 560
C on several successive
days
Inspissation
Media containing egg or serum such as Lowenstein-Jensen
and Loeffler’s serum slope are rendered sterile by heating
at 80-850
C for 30 min on three successive days
This process is called inspissation and instrument used is
called inspissator
LTSF sterilization
Used for sterilizing items which cannot withstand the temperatue
of 1000
C
In this method steam at 750
C with formaldehyde vapor is used
Inspissator
Inspissator Water bath
Water bath
Temperature at 100°C
Temperature at 100°C
1.
1. Boiling
Boiling
2.
2. Tyndallisation
Tyndallisation
3.
3. Steam
Steam
sterilisation
sterilisation
Boiling at 1000
C
 Boiling at 1000
C for 10-30 min kills all vegetative bacteria and
some bacterial spores
 Sporing bacteria required prolonged periods of boiling
 Therefore, it is not recommended for sterilization of
surgical instruments
 Addition of 2% sodium bicarbonate may promote sterilization
Uses
 For the disinfection of medical and surgical equipment – when
sterility is not essential in emergency or under field conditions
Free steam at 1000
C
 Steam at normal atmosheric pressure is at 1000
C
 Used to sterilize heat-labile culture media
 A Koch or Arnold steam sterilizer is used
 It consists of a vertical metal cylinder with a removable
conical lid
 Single exposure to steam for 90 min ensures complete
sterilization
Tyndallisation
An exposure of steam1000
C for 20 min on three consecutive
days is known as Tyndallization or intermittent sterilisation
The instrument used is Koch or Arnold steam sterilizer
Principle
First exposure kills all the vegetative forms, and in the intervals
between the heatings the remaining spores germinate into
vegetative forms which are killed on subsequent heating
Uses
Used for sterilisation of egg, serum or sugar containing media
Koch or Arnold steam sterilizer
Temperature above 1000
C
Steam under pressure
Saturated steam is more efficient sterilizing agent than hot air
because
1. It provides greater lethal action of moist heat
2. It is quicker in heating up the exposed articles
3. It can easily penetrate porous material
4. When the steam meets the cooler surface of the article,
it condenses into a small volume of water and liberates
considerable latent heat
Autoclave
Principle
Water boils when its vapor pressure equals that of the surrounding
atmosphere
When pressure inside a closed vessel increases, the temperature
at which water boils also increases
Saturated steam has penetrative power and is a better sterilizing
agent than dry heat
Steam condenses to water and gives up its latent heat to
the surface when it comes into contact with a cooler surface
The large reduction in volume, sucks in more steam to the area
and the process continues till the temperature of that surface
is raised to that of the steam
Components of autoclave
Components of autoclave:
:
 Consists of vertical or horizontal cylinder of
Consists of vertical or horizontal cylinder of
gunmetal or stainless steel.
gunmetal or stainless steel.
 Lid is fastened by screw clamps and rendered
Lid is fastened by screw clamps and rendered
air tight by an asbestos washer.
air tight by an asbestos washer.
 Lid bears a discharge tap for air and steam, a
Lid bears a discharge tap for air and steam, a
pressure gauge and a safety valve.
pressure gauge and a safety valve.
Holding period
Holding period
121
1210
0
C for 15 min (15 lbs or psi pressure)
C for 15 min (15 lbs or psi pressure)
Precautions
1. All the air must be removed from the autoclave chamber
The admixture of air with steam results in low temperature
being achieved
Air hinders penetration of steam
The air being denser forms a cooler layer in the lower part of
the autoclave
2. Materials should be arranged in such a manner which ensures
free circulation of steam inside the chamber
3. Lid should not open until inside pressure reaches to the
atmospheric pressure
 Uses :
Uses :
1.
1. To sterilize culture media,
To sterilize culture media,
rubber material, gowns,
rubber material, gowns,
dressings, gloves, instruments
dressings, gloves, instruments
and pharmaceutical products
and pharmaceutical products
2.
2. For all materials that are water
For all materials that are water
containing, permeable or
containing, permeable or
wettable and not liable to be
wettable and not liable to be
damaged by the process
damaged by the process
3.
3. Useful for materials which
Useful for materials which
cannot withstand the high
cannot withstand the high
temperature of hot air oven
temperature of hot air oven
Sterilisation controls
Sterilisation controls
1.
1. Thermocouples
Thermocouples
2.
2. Bacterial spores-
Bacterial spores-
Bacillus
Bacillus
stearothermophilus
stearothermophilus
3.
3. Browne’s tube
Browne’s tube
4.
4. Autoclave tapes
Autoclave tapes
Filtration
Filtration
Sterilize solutions that may be
damaged or denatured by high
temperatures or chemical
agents
Used for the sterilization of heat
labile materials such as sera,
sugar solutions, and antibiotics
Types of Filters
Types of Filters
1. Earthenware filters
1. Earthenware filters
(Candle filters)
(Candle filters)
2. Asbestos disc
2. Asbestos disc
(Seitz) filters
(Seitz) filters
3. Sintered glass filters
3. Sintered glass filters
4. Membrane filters
4. Membrane filters
5. Syringe filters
5. Syringe filters
6. Air filters
6. Air filters
Earthenware filters
Manufactured in several different grades of porosity
Used widely for purification of water for industrial
and drinking purposes
They are of two types
1. Unglazed ceramic filters
eg: Chamberland and Doulton filters
2. Compressed diatomaceous earth filters
eg: Berkefeld and Mandler filters
Earthenware (Candle) filters
Asbestos filters
 Made up of a disc of asbestos (magnesium trisilicate)
 Discs are available with different grades of porosity
 It is supported on a perforated metal disc within a metal funnel
 It is then fitted onto a sterile flask through a silicone rubber
bung
 The fluid to be sterilized is put into the funnel and flask
connected to the exhaust pump through its side tap
Asbestos Filter holder
Asbestos Filter holder
Sintered glass filters
Prepared by fusing finely powdered glass particles
Available in different pore sizes
Pore size can be controlled by the general particle size of
the glass powder
The filters are easily cleaned, have low absorption properties
and do not shed particles
But they are fragile and relatively expensive
Sintered glass filter
Sintered glass filter
Membrane filters
Made of variety of polymeric materials such as cellulose nitrate,
cellulose diacetate, polycarbonate and polyester
Membrane filters are available in pore sizes of 0.015 to 12 µm
The 0.22 µm filter is most commonly used because the pore size
is smaller than that of bacteria
These are routinely used in water analysis, bacterial counts of
water, sterility testing, and for the preparation of solutions for
parenteral use
Membrane filters
Membrane filters
Syringe filters
Syringes fitted with membrane filters of different pore sizes
are available
For sterilization, the fluid is forced through the the disc
(membrane) by pressing the piston of the syringe
Air filters
Air can also be sterilized by filtration
Large volumes of air may be rapidly freed from infection by
passage through high efficiency particulate air (HEPA) filters
They are used in laminar air flow system in microbiology
laboratories
HEPA filters can remove particles of 0.3 µm or larger
The roles of HEPA filters in biological flow
The roles of HEPA filters in biological flow
safety cabinets
safety cabinets
Exhaust HEPA
filter
Blower
Supply HEPA
filter
Light
High-velocity
air barrier
Safety glass
viewscreen
03/09/14 Dr.T.V.Rao MD
53
Radiation
Two types of radiations are used for sterilization
1.Non-ionising
2.Ionising
Non-ionising radiations
These inculde infrared and ultraviolet (UV) radiations
Infrared is used for rapid mass sterilisation of syringes
and catheters
UV radiation with wavelength of 240-280 nm has marked
bactericidal activity
 It acts by denaturation of bacterial protein and interference
with DNA replication (produces thymine dimers)
 They can penetrate only a few mm into liquids and not at
all into solids
 UV radiation is used for disinfecting enclosed areas such as
bacteriological laboratory, inoculation hoods, laminar flow
and operation theatres
 Most vegetative bacteria are susceptible but spores are highly
resistant
 Susceptibility of viruses is variable
 Source of UV radiations must be shielded otherwise causes
damage to skin and eyes.
Ionising radiations
 These include X-rays, Υ (gamma) rays and cosmic rays
Possess high penetrative power and are highly lethal to all
cells including bacteria
They damage DNA by various mechanisms
Gamma radiations are used for sterilization of disposable
items such as plastic syringes, swabs, culture plates,
cannulas, catheters etc
 Since theres is no appreciable increase in the temperature,
in this method it is known as “cold sterilisation”
 Large commercial plants use gamma radiation emitted from
a radioactive element, usually cobalt 60
 The advantage of this method include speed, high penetrating
power (it can sterilise materials through outer packages
and wrappings)
 Bacillus pumilis used to test the efficacy of ionizing radiations
Chemical methods
A variety of chemical agents are used as antiseptics
and disinfectants. An ideal antiseptic or disinfectant should
1) Be fast acting in presence of organic substances
2) Be effective against all types of infectious agents without
destroying tissue or acting as a poison if ingested
3) Easily penetrate material to be disinfected, without
damaging/discoloring it
4) Be easy to prepare, stable when exposed to light, heat or
other environ-mental factors
5) Be inexpensive, easy to obtain and use
6) Not have an unpleasant odor
Chemical agents act in various ways. The main modes of action
are
1. Protein coagulation
2. Distruption of cell membrane
3. Removal of free sulphydryl groups
4. Substrate competition
Factors that determine the potency of
disinfectants are
a) Concentration of the substance
b) Time of action
c) pH of the medium
d) Temperature
e) Nature of organism
f) Presence of organic matter
Disinfectants can be divided into three groups
1.High level disinfectants
(Glutaraldehyde, hydrogen peroxide, peracetic acid
and chlorine compounds)
2. Intermediate level disinfectants
(Alcohol, iodophores and phenolic compounds)
3. Low level disinfectants
(Quarternary ammonium compounds)
Alcohols
 Ethanol and isopropanol are the most frequently used
 Used as skin antiseptics and act by denaturing bacterial
proteins
 Rapidly kill bacteria including tubercle bacilli but they have
no sporicidal or virucidal activity
 60-70% is most effective
 Isopropyl alcohol is preferred to ethyl alcohol as it is a better
fat solvent, more bactericidal and less volatile
 Methyl alcohol is effective against fungal spores
Aldehydes
Two aldehydes (formaldehyde and glutaraldehyde) are
currently of considerable importance
Formaldehyde
 Formaldehyde is active against the aminogroup in the protein
molecules
 It is lethal to bacteria and their spores, viruses and fungi
 It is employed in the liquid and vapor states
 A 10% aqueous solution is routinely used
Uses
To sterilise bacterial vaccines
10% formalin containing 0.5% sodium tetraborate is used to
sterilize clean metal instruments
Formaldehyde gas is used for sterilizing instruments, heat
sensitive catheters and for fumigating wards, sick rooms
and laboratories
Glutaraldehyde
Action similar to formaldehyde
More active and less toxic than formaldehyde
It is used as 2% buffered solution
It is available commercially as ‘cidex’
Uses
For sterilization of cystoscopes, endoscopes and
bronchoscopes
To sterilize plastic endotracheal tubes, face masks,
corrugated rubber anaesthetic tubes and metal instruments
Phenols
Obtained by distillation of coal tar between temperatures of
1700
C and 2700
C
Lethal effect is due to cell membrane damage
Phenol (1%) has bactericidal action
Phenol derivatives like cresol, chlorhexidine and hexachloro-
phane are commonly used as antiseptics
Cresols
Lysol is a solution of cresols in soap
Most commonly used for sterilization of infected glasswares,
cleaning floors, disinfection of excreta
Chlorhexidine
Savlon (Chlorhexidine and cetrimide) is widely used in wounds,
pre-operative disinfection of skin
More active against Gram positive than Gram negative bacteria
No action against tubercle bacilli or spores and have very
little activity against viruses
Has a good fungicidal activity
Halogens
Chlorine and iodine are two commonly used disinfectants
They are bactericidal and are effective against sporing bacteria
and viruses
Chlorine is used in water supplies, swimming pools, food and
dairy industries
Chlorine is used in the form of bleaching powder, sodium-
hypochlorite and chloramine
Hypochlorites have a bactericidal, fungicidal, virucidal and sporicidal
action
Bleaching powder or hypochlorite solution are the most widely used
for HIV infected material
Chloramines are used as antiseptics for dressing wounds
Iodine
Iodine in aqueous and alcoholic solution used as skin disinfectant
Iodine often has been applied as tincture of iodine (2% iodine in a
water-ethanol solution of potassium iodide)
Actively bactericidal, moderate action against spores
Also active against the tubercle bacteria and viruses
Compounds of iodine with surface active agents known
as iodophores
Used in hospitals for preoperative skin degerming
Povidine-iodine (Betadine) for wounds and Wescodyne for skin
and laboratory disinfection are some examples of iodophores
Dyes
Aniline and acridine dyes are used extensively as skin and wound
antiseptics
Aniline dyes include crystal violet, brilliant green, and malachite green
are more active against gram positive organisms
No activity against tubercle bacilli
They interfere with the synthesis of peptidoglycan of the cell wall
Their activity is inhibited by organic material such as pus
Acridine dyes also more active against gram positive organisms
Acridine dyes affected very little by the presence of organic matter
More important dyes are proflavine, acriflavine, euflavine
and aminacrine
They interfere with the synthesis of nucleic acids and proteins
in bacterial cells
Metallic salts
Salts of silver, copper and mercury are used as disinfectants
Protein coagulants and have the capacity to combine with free
sulphydryl groups
The organic compounds thiomersal, phenyl mercury nitrate and
mercurochrome are less toxic and are used as mild antisepics
Copper salts are used as fungicides
Surface active agents
Substances which alter energy relationships at interfaces,
producing a reduction of surface tension; 4 types
1.Anionic
Common soaps, have strong detergent but weak
antimicrobial properties
2. Cationic
Act on phosphate group of the cell membrane
Eg: Quaternary ammonium compounds such as benzalkonium
chloride and acetyl trimethyl ammonium bromide (cetrimide)
3. Nonionic
4. Amphoteric (Tego compounds)
Vapour Phase Disinfectants
Formaldehyde gas
Employed for fumigation of heat-sensitive equipment (anaesthetic
machine and baby incubators), operation theatres, wards and
laboratories etc
Formaldehyde gas is generated by adding 150 gm of KMnO4 to
280 ml of formalin for 1000 cubic feet of room volume
This reaction produces considerable heat and so heat resistant
containers should be used
Sterilisation is achieved by condensation of gas on exposed
surfaces
After completion of disinfection, the effect of irritant vapours
should be nullified by exposure to ammonia vapour
Ethylene oxide (ETO)
Colourless liquid with a boiling point of 10.70
C
Highly lethal to all kinds of microbes including spores
Action is due to its alkylating the amino, carboxyl, hydroxyl and
sulphydryl groups in protein molecules
In addition it reacts with DNA and RNA
Highly inflammable and in concentrations (>3%) highly explosive
By mixing with inert gases such as CO2, its explosive tendency
can be eliminated
Uses
Used for sterilising plastic and rubber articles, respirators,
heart-lung machines, sutures, dental equipments and
clothing
It is commercially used to sterilise disposable plastic syringes,
petridishes etc
It has a high penetrating power and thus can sterilise
prepackaged materials
Bacillus globigi ( a red pigmented variant of B. subtilis) has
been used as a biological control fot testing of ETO sterilisers
Betapropiolactone (BPL)
Condensation product of ketane and formaldehyde
Boiling point: 1630
C
Has low penetrating power but has a rapid action
For sterilization of biological products 0.2% BPL is used
Capable of killing all microorganisms and is very active against
viruses
Uses
In the liquid form it has been used to sterilize vaccines and sera
Recent vapor phase disinfectants
Hydrogen peroxide
Used to decontaminate biological safety cabinets
Peracetic acid
It ia an oxidising agent
One of the high level disinfectants
Used in plasma sterilizers
Testing of disinfectants
1. Minimum inhibitory concentration (MIC)
2. Rideal Walker test
3. Chick Martin test
4. Capacity test (Kelsey and Sykes test)
5. In-use test
Sterilisation of prions
Dry heat
3600
C for one hour
Moist heat
134-1380
C for 18 min
Chemicals
25% sodium hypochlorite for one hour
Sensitive to household bleach, phenol (90%) and iodine
disinfectants
Thank you !!!
Thank you !!!

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  • 1. Sterilisation and Sterilisation and Disinfection Disinfection Dr. Pendru Raghunath Reddy Dr. Pendru Raghunath Reddy
  • 2. Why we need Sterilization? Why we need Sterilization?  Microorganisms capable of causing infection are Microorganisms capable of causing infection are constantly present in the external environment and on constantly present in the external environment and on the human body the human body  Microorganisms are responsible for contamination and Microorganisms are responsible for contamination and infection infection  The aim of sterilisation is to remove or destroy them The aim of sterilisation is to remove or destroy them from materials or from surfaces from materials or from surfaces
  • 3. Definitions: Definitions: Sterilisation : Sterilisation : – It is a process by which an article, surface or It is a process by which an article, surface or medium is made free of all microorganisms either medium is made free of all microorganisms either in vegetative or spore form in vegetative or spore form Disinfection : Disinfection : – Destruction of all pathogens or organisms capable Destruction of all pathogens or organisms capable of producing infections but not necessarily spores. of producing infections but not necessarily spores. – All organisms may not be killed but the number is All organisms may not be killed but the number is reduced to a level that is no longer harmful to reduced to a level that is no longer harmful to health. health.
  • 4. Antiseptics : Antiseptics :  Chemical disinfectants which can safely applied to Chemical disinfectants which can safely applied to living tissues and are used to prevent infection by living tissues and are used to prevent infection by inhibiting the growth of microorganisms inhibiting the growth of microorganisms Asepsis : Asepsis :  Technique by which the occurrence of infection into Technique by which the occurrence of infection into an uninfected tissue is prevented. an uninfected tissue is prevented.
  • 6. Physical methods: Physical methods:  Physical Physical methods: methods: 1. 1. Sunlight Sunlight 2. 2. Drying Drying 3. 3. Heat Heat 1. 1. Dry heat Dry heat 2. 2. Moist heat Moist heat 4. 4. Filtration Filtration 5. 5. Radiation Radiation 6
  • 7. Chemical methods Chemical methods • Chemical methods: Chemical methods: 1. 1. Alcohols Alcohols 2. 2. Aldehydes Aldehydes 3. 3. Phenols Phenols 4. 4. Halogens Halogens 5. 5. Oxidizing agents Oxidizing agents 6. 6. Salts Salts 7. 7. Surface active agents Surface active agents 8. 8. Dyes Dyes 9. 9. Vapor phase disinfectants Vapor phase disinfectants
  • 8. Physical methods 1. Sunlight Sunlight possesses appreciable bactericidal activity Due to its content of ultraviolet rays & heat rays Under natural conditions, its sterilising power varies according to circumstances Natural method of sterilisation of water in tanks, rivers and lakes 2. Drying Moisture is essential for the growth of bacteria Drying in air has a deleterious effect on many bacteria This method is unreliable and is only of theoretical interest
  • 9. 3. Heat Most reliable method of sterilisation The factors influencing sterilisation by heat are: Nature of heat Temperature and time Number of microorganisms present Characteristics of the organisms, such as species, strain and sporing capacity Type of material from which the organisms have to be eradicated
  • 10. Mechanism of action Dry heat Kills organisms by protein denaturation, oxidative damage and toxic effects of elevated levels of electrolytes Moist heat Kills microorganisms by coagulation and denaturation of their enzymes and structural proteins
  • 11.  Dry heat: Dry heat: 1. 1. Red heat Red heat 2. 2. Flaming Flaming 3. 3. Incineration Incineration 4. 4. Hot air oven Hot air oven
  • 12. Red heat Red heat Materials are held in Materials are held in the flame of a bunsen the flame of a bunsen burner till they become burner till they become red hot. red hot.  Inoculating wires or Inoculating wires or loops loops  Tips of forceps Tips of forceps  Surface of searing Surface of searing spatulae spatulae  Needles Needles
  • 13. Flaming Flaming Materials are passed Materials are passed through the flame of a through the flame of a bunsen burner without bunsen burner without allowing them to allowing them to become red hot. become red hot.  Glass slides Glass slides  scalpels scalpels  Mouths of culture Mouths of culture tubes and bottles tubes and bottles
  • 14. Incineration: Incineration: Materials are reduced Materials are reduced to ashes by burning. to ashes by burning. Instrument used was Instrument used was incinerator. incinerator. Soiled dressings Soiled dressings Animal carcasses Animal carcasses Bedding Bedding Pathological material Pathological material
  • 15. Hot air oven Most widely used method of sterilisation by dry heat It is used to process materials which can withstand high temperatures, but which are likely to be affected by contact with steam It is a method of choice for sterilisation of glassware, forceps, scissors, scalpels, swab sticks packed in test tubes Materials such as oils, jellies and powders which are impervious to steam are sterilised by hot air oven Hot air oven is electrically heated and is fitted with a termostat that maintains the chamber air at a chosen temperature Fitted with a fan that distributes hot air in the chamber
  • 17.
  • 18. Holding temperature & time General purpose Temperature and time: 1600 C for 2 hours 1700 C for 1 hour 1800 C for 30 minutes Cutting instruments such as those used in ophthalmic surgery, Should be sterilized at 1500 C for 2 hours Oils, glycerol and dusting powder should be sterilised at 1500 C for 1 hour
  • 19. Precautions Precautions 1. 1. Should not be overloaded Should not be overloaded 2. 2. Arranged in a manner which allows free circulation of Arranged in a manner which allows free circulation of air air 3. 3. Material to be sterilized should be perfectly dry. Material to be sterilized should be perfectly dry. 4. 4. Test tubes, flasks etc. should be fitted with cotton Test tubes, flasks etc. should be fitted with cotton plugs. plugs. 5. 5. petridishes and pipetts should be wrapped in paper. petridishes and pipetts should be wrapped in paper. 6. 6. Rubber materials and inflammable materials should Rubber materials and inflammable materials should not be kept inside. not be kept inside. 7. 7. The oven must be allowed to cool for two hours The oven must be allowed to cool for two hours before opening, since glass ware may crack by before opening, since glass ware may crack by sudden cooling. sudden cooling.
  • 20. Uses of Hot Air Oven Uses of Hot Air Oven  Sterilisation of Sterilisation of 1. 1. Glassware like glass syringes, petri Glassware like glass syringes, petri dishes, pipettes and test tubes. dishes, pipettes and test tubes. 2. 2. Surgical instruments like scalpels, Surgical instruments like scalpels, scissors, forceps etc. scissors, forceps etc. 3. 3. Chemicals like liquid paraffin, fats etc. Chemicals like liquid paraffin, fats etc.
  • 21. Sterilisation controls Sterilisation controls 1. 1. Spores of Spores of Bacillus Bacillus subtilis subsp. Niger subtilis subsp. Niger 2. 2. Thermocouples Thermocouples 3. 3. Browne’s tube Browne’s tube (Tube containing red (Tube containing red colour solution is inserted colour solution is inserted in each load and a colour in each load and a colour change from red to green change from red to green indicates proper indicates proper sterilization) sterilization)
  • 22. Moist heat Moist heat is divided into three forms 1.Temperature below 1000 C 2.At a temperature of 1000 C 3.Temperature above 1000 C Temperatures below 1000 C 1. Pasteurization 2. Vaccine bath 3. Water bath 4. Inspissation 5. Low temperature steam-formaldehyde (LTSF) sterilization
  • 23. Pasteurization  Milk is sterilised by this method; Two methods 1.Holder method (630 C for 30 min followed by rapid cooling to 130 C or lower) 2. Flash method (720 C for 15-20 seconds followed by rapid cooling to 130 C or lower) The dairy industry sometimes uses ultrahightemperature (UHT) sterilization (140 to 1500 C for 1-3 seconds followed by rapid cooling to 130 C or lower) All nonsporing pathogens such as mycobacteria, brucellae and salmonellae are destroyed by these processes Coxiella burnetii is relatively heat resistant and may survive the holder method
  • 24. Principle of Pasteurization Principle of Pasteurization 03/09/14 Dr.T.V.Rao MD 24
  • 25. Vaccine bath Vaccines prepared from nonsporing bacteria may be inactivated in a water bath at 600 C for 1 hour Water bath Serum or body fluids containing coagulable proteins can be sterilized by heating for 1 hour at 560 C on several successive days
  • 26. Inspissation Media containing egg or serum such as Lowenstein-Jensen and Loeffler’s serum slope are rendered sterile by heating at 80-850 C for 30 min on three successive days This process is called inspissation and instrument used is called inspissator LTSF sterilization Used for sterilizing items which cannot withstand the temperatue of 1000 C In this method steam at 750 C with formaldehyde vapor is used
  • 28. Temperature at 100°C Temperature at 100°C 1. 1. Boiling Boiling 2. 2. Tyndallisation Tyndallisation 3. 3. Steam Steam sterilisation sterilisation
  • 29. Boiling at 1000 C  Boiling at 1000 C for 10-30 min kills all vegetative bacteria and some bacterial spores  Sporing bacteria required prolonged periods of boiling  Therefore, it is not recommended for sterilization of surgical instruments  Addition of 2% sodium bicarbonate may promote sterilization Uses  For the disinfection of medical and surgical equipment – when sterility is not essential in emergency or under field conditions
  • 30. Free steam at 1000 C  Steam at normal atmosheric pressure is at 1000 C  Used to sterilize heat-labile culture media  A Koch or Arnold steam sterilizer is used  It consists of a vertical metal cylinder with a removable conical lid  Single exposure to steam for 90 min ensures complete sterilization
  • 31. Tyndallisation An exposure of steam1000 C for 20 min on three consecutive days is known as Tyndallization or intermittent sterilisation The instrument used is Koch or Arnold steam sterilizer Principle First exposure kills all the vegetative forms, and in the intervals between the heatings the remaining spores germinate into vegetative forms which are killed on subsequent heating Uses Used for sterilisation of egg, serum or sugar containing media
  • 32. Koch or Arnold steam sterilizer
  • 33. Temperature above 1000 C Steam under pressure Saturated steam is more efficient sterilizing agent than hot air because 1. It provides greater lethal action of moist heat 2. It is quicker in heating up the exposed articles 3. It can easily penetrate porous material 4. When the steam meets the cooler surface of the article, it condenses into a small volume of water and liberates considerable latent heat
  • 34. Autoclave Principle Water boils when its vapor pressure equals that of the surrounding atmosphere When pressure inside a closed vessel increases, the temperature at which water boils also increases Saturated steam has penetrative power and is a better sterilizing agent than dry heat Steam condenses to water and gives up its latent heat to the surface when it comes into contact with a cooler surface The large reduction in volume, sucks in more steam to the area and the process continues till the temperature of that surface is raised to that of the steam
  • 35. Components of autoclave Components of autoclave: :  Consists of vertical or horizontal cylinder of Consists of vertical or horizontal cylinder of gunmetal or stainless steel. gunmetal or stainless steel.  Lid is fastened by screw clamps and rendered Lid is fastened by screw clamps and rendered air tight by an asbestos washer. air tight by an asbestos washer.  Lid bears a discharge tap for air and steam, a Lid bears a discharge tap for air and steam, a pressure gauge and a safety valve. pressure gauge and a safety valve. Holding period Holding period 121 1210 0 C for 15 min (15 lbs or psi pressure) C for 15 min (15 lbs or psi pressure)
  • 36.
  • 37.
  • 38. Precautions 1. All the air must be removed from the autoclave chamber The admixture of air with steam results in low temperature being achieved Air hinders penetration of steam The air being denser forms a cooler layer in the lower part of the autoclave 2. Materials should be arranged in such a manner which ensures free circulation of steam inside the chamber 3. Lid should not open until inside pressure reaches to the atmospheric pressure
  • 39.  Uses : Uses : 1. 1. To sterilize culture media, To sterilize culture media, rubber material, gowns, rubber material, gowns, dressings, gloves, instruments dressings, gloves, instruments and pharmaceutical products and pharmaceutical products 2. 2. For all materials that are water For all materials that are water containing, permeable or containing, permeable or wettable and not liable to be wettable and not liable to be damaged by the process damaged by the process 3. 3. Useful for materials which Useful for materials which cannot withstand the high cannot withstand the high temperature of hot air oven temperature of hot air oven
  • 40. Sterilisation controls Sterilisation controls 1. 1. Thermocouples Thermocouples 2. 2. Bacterial spores- Bacterial spores- Bacillus Bacillus stearothermophilus stearothermophilus 3. 3. Browne’s tube Browne’s tube 4. 4. Autoclave tapes Autoclave tapes
  • 41. Filtration Filtration Sterilize solutions that may be damaged or denatured by high temperatures or chemical agents Used for the sterilization of heat labile materials such as sera, sugar solutions, and antibiotics
  • 42. Types of Filters Types of Filters 1. Earthenware filters 1. Earthenware filters (Candle filters) (Candle filters) 2. Asbestos disc 2. Asbestos disc (Seitz) filters (Seitz) filters 3. Sintered glass filters 3. Sintered glass filters 4. Membrane filters 4. Membrane filters 5. Syringe filters 5. Syringe filters 6. Air filters 6. Air filters
  • 43. Earthenware filters Manufactured in several different grades of porosity Used widely for purification of water for industrial and drinking purposes They are of two types 1. Unglazed ceramic filters eg: Chamberland and Doulton filters 2. Compressed diatomaceous earth filters eg: Berkefeld and Mandler filters
  • 45. Asbestos filters  Made up of a disc of asbestos (magnesium trisilicate)  Discs are available with different grades of porosity  It is supported on a perforated metal disc within a metal funnel  It is then fitted onto a sterile flask through a silicone rubber bung  The fluid to be sterilized is put into the funnel and flask connected to the exhaust pump through its side tap
  • 47. Sintered glass filters Prepared by fusing finely powdered glass particles Available in different pore sizes Pore size can be controlled by the general particle size of the glass powder The filters are easily cleaned, have low absorption properties and do not shed particles But they are fragile and relatively expensive
  • 49. Membrane filters Made of variety of polymeric materials such as cellulose nitrate, cellulose diacetate, polycarbonate and polyester Membrane filters are available in pore sizes of 0.015 to 12 µm The 0.22 µm filter is most commonly used because the pore size is smaller than that of bacteria These are routinely used in water analysis, bacterial counts of water, sterility testing, and for the preparation of solutions for parenteral use
  • 51. Syringe filters Syringes fitted with membrane filters of different pore sizes are available For sterilization, the fluid is forced through the the disc (membrane) by pressing the piston of the syringe
  • 52. Air filters Air can also be sterilized by filtration Large volumes of air may be rapidly freed from infection by passage through high efficiency particulate air (HEPA) filters They are used in laminar air flow system in microbiology laboratories HEPA filters can remove particles of 0.3 µm or larger
  • 53. The roles of HEPA filters in biological flow The roles of HEPA filters in biological flow safety cabinets safety cabinets Exhaust HEPA filter Blower Supply HEPA filter Light High-velocity air barrier Safety glass viewscreen 03/09/14 Dr.T.V.Rao MD 53
  • 54. Radiation Two types of radiations are used for sterilization 1.Non-ionising 2.Ionising Non-ionising radiations These inculde infrared and ultraviolet (UV) radiations Infrared is used for rapid mass sterilisation of syringes and catheters UV radiation with wavelength of 240-280 nm has marked bactericidal activity
  • 55.  It acts by denaturation of bacterial protein and interference with DNA replication (produces thymine dimers)  They can penetrate only a few mm into liquids and not at all into solids  UV radiation is used for disinfecting enclosed areas such as bacteriological laboratory, inoculation hoods, laminar flow and operation theatres  Most vegetative bacteria are susceptible but spores are highly resistant  Susceptibility of viruses is variable  Source of UV radiations must be shielded otherwise causes damage to skin and eyes.
  • 56. Ionising radiations  These include X-rays, Υ (gamma) rays and cosmic rays Possess high penetrative power and are highly lethal to all cells including bacteria They damage DNA by various mechanisms Gamma radiations are used for sterilization of disposable items such as plastic syringes, swabs, culture plates, cannulas, catheters etc
  • 57.  Since theres is no appreciable increase in the temperature, in this method it is known as “cold sterilisation”  Large commercial plants use gamma radiation emitted from a radioactive element, usually cobalt 60  The advantage of this method include speed, high penetrating power (it can sterilise materials through outer packages and wrappings)  Bacillus pumilis used to test the efficacy of ionizing radiations
  • 58. Chemical methods A variety of chemical agents are used as antiseptics and disinfectants. An ideal antiseptic or disinfectant should 1) Be fast acting in presence of organic substances 2) Be effective against all types of infectious agents without destroying tissue or acting as a poison if ingested 3) Easily penetrate material to be disinfected, without damaging/discoloring it 4) Be easy to prepare, stable when exposed to light, heat or other environ-mental factors 5) Be inexpensive, easy to obtain and use 6) Not have an unpleasant odor
  • 59. Chemical agents act in various ways. The main modes of action are 1. Protein coagulation 2. Distruption of cell membrane 3. Removal of free sulphydryl groups 4. Substrate competition Factors that determine the potency of disinfectants are a) Concentration of the substance b) Time of action c) pH of the medium d) Temperature e) Nature of organism f) Presence of organic matter
  • 60. Disinfectants can be divided into three groups 1.High level disinfectants (Glutaraldehyde, hydrogen peroxide, peracetic acid and chlorine compounds) 2. Intermediate level disinfectants (Alcohol, iodophores and phenolic compounds) 3. Low level disinfectants (Quarternary ammonium compounds)
  • 61. Alcohols  Ethanol and isopropanol are the most frequently used  Used as skin antiseptics and act by denaturing bacterial proteins  Rapidly kill bacteria including tubercle bacilli but they have no sporicidal or virucidal activity  60-70% is most effective  Isopropyl alcohol is preferred to ethyl alcohol as it is a better fat solvent, more bactericidal and less volatile  Methyl alcohol is effective against fungal spores
  • 62. Aldehydes Two aldehydes (formaldehyde and glutaraldehyde) are currently of considerable importance Formaldehyde  Formaldehyde is active against the aminogroup in the protein molecules  It is lethal to bacteria and their spores, viruses and fungi  It is employed in the liquid and vapor states  A 10% aqueous solution is routinely used
  • 63. Uses To sterilise bacterial vaccines 10% formalin containing 0.5% sodium tetraborate is used to sterilize clean metal instruments Formaldehyde gas is used for sterilizing instruments, heat sensitive catheters and for fumigating wards, sick rooms and laboratories
  • 64. Glutaraldehyde Action similar to formaldehyde More active and less toxic than formaldehyde It is used as 2% buffered solution It is available commercially as ‘cidex’ Uses For sterilization of cystoscopes, endoscopes and bronchoscopes To sterilize plastic endotracheal tubes, face masks, corrugated rubber anaesthetic tubes and metal instruments
  • 65. Phenols Obtained by distillation of coal tar between temperatures of 1700 C and 2700 C Lethal effect is due to cell membrane damage Phenol (1%) has bactericidal action Phenol derivatives like cresol, chlorhexidine and hexachloro- phane are commonly used as antiseptics Cresols Lysol is a solution of cresols in soap Most commonly used for sterilization of infected glasswares, cleaning floors, disinfection of excreta
  • 66. Chlorhexidine Savlon (Chlorhexidine and cetrimide) is widely used in wounds, pre-operative disinfection of skin More active against Gram positive than Gram negative bacteria No action against tubercle bacilli or spores and have very little activity against viruses Has a good fungicidal activity
  • 67. Halogens Chlorine and iodine are two commonly used disinfectants They are bactericidal and are effective against sporing bacteria and viruses Chlorine is used in water supplies, swimming pools, food and dairy industries Chlorine is used in the form of bleaching powder, sodium- hypochlorite and chloramine Hypochlorites have a bactericidal, fungicidal, virucidal and sporicidal action Bleaching powder or hypochlorite solution are the most widely used for HIV infected material Chloramines are used as antiseptics for dressing wounds
  • 68. Iodine Iodine in aqueous and alcoholic solution used as skin disinfectant Iodine often has been applied as tincture of iodine (2% iodine in a water-ethanol solution of potassium iodide) Actively bactericidal, moderate action against spores Also active against the tubercle bacteria and viruses Compounds of iodine with surface active agents known as iodophores Used in hospitals for preoperative skin degerming Povidine-iodine (Betadine) for wounds and Wescodyne for skin and laboratory disinfection are some examples of iodophores
  • 69. Dyes Aniline and acridine dyes are used extensively as skin and wound antiseptics Aniline dyes include crystal violet, brilliant green, and malachite green are more active against gram positive organisms No activity against tubercle bacilli They interfere with the synthesis of peptidoglycan of the cell wall Their activity is inhibited by organic material such as pus Acridine dyes also more active against gram positive organisms Acridine dyes affected very little by the presence of organic matter
  • 70. More important dyes are proflavine, acriflavine, euflavine and aminacrine They interfere with the synthesis of nucleic acids and proteins in bacterial cells Metallic salts Salts of silver, copper and mercury are used as disinfectants Protein coagulants and have the capacity to combine with free sulphydryl groups The organic compounds thiomersal, phenyl mercury nitrate and mercurochrome are less toxic and are used as mild antisepics Copper salts are used as fungicides
  • 71. Surface active agents Substances which alter energy relationships at interfaces, producing a reduction of surface tension; 4 types 1.Anionic Common soaps, have strong detergent but weak antimicrobial properties 2. Cationic Act on phosphate group of the cell membrane Eg: Quaternary ammonium compounds such as benzalkonium chloride and acetyl trimethyl ammonium bromide (cetrimide) 3. Nonionic 4. Amphoteric (Tego compounds)
  • 72. Vapour Phase Disinfectants Formaldehyde gas Employed for fumigation of heat-sensitive equipment (anaesthetic machine and baby incubators), operation theatres, wards and laboratories etc Formaldehyde gas is generated by adding 150 gm of KMnO4 to 280 ml of formalin for 1000 cubic feet of room volume This reaction produces considerable heat and so heat resistant containers should be used Sterilisation is achieved by condensation of gas on exposed surfaces After completion of disinfection, the effect of irritant vapours should be nullified by exposure to ammonia vapour
  • 73. Ethylene oxide (ETO) Colourless liquid with a boiling point of 10.70 C Highly lethal to all kinds of microbes including spores Action is due to its alkylating the amino, carboxyl, hydroxyl and sulphydryl groups in protein molecules In addition it reacts with DNA and RNA Highly inflammable and in concentrations (>3%) highly explosive By mixing with inert gases such as CO2, its explosive tendency can be eliminated
  • 74. Uses Used for sterilising plastic and rubber articles, respirators, heart-lung machines, sutures, dental equipments and clothing It is commercially used to sterilise disposable plastic syringes, petridishes etc It has a high penetrating power and thus can sterilise prepackaged materials Bacillus globigi ( a red pigmented variant of B. subtilis) has been used as a biological control fot testing of ETO sterilisers
  • 75. Betapropiolactone (BPL) Condensation product of ketane and formaldehyde Boiling point: 1630 C Has low penetrating power but has a rapid action For sterilization of biological products 0.2% BPL is used Capable of killing all microorganisms and is very active against viruses Uses In the liquid form it has been used to sterilize vaccines and sera
  • 76. Recent vapor phase disinfectants Hydrogen peroxide Used to decontaminate biological safety cabinets Peracetic acid It ia an oxidising agent One of the high level disinfectants Used in plasma sterilizers
  • 77. Testing of disinfectants 1. Minimum inhibitory concentration (MIC) 2. Rideal Walker test 3. Chick Martin test 4. Capacity test (Kelsey and Sykes test) 5. In-use test
  • 78. Sterilisation of prions Dry heat 3600 C for one hour Moist heat 134-1380 C for 18 min Chemicals 25% sodium hypochlorite for one hour Sensitive to household bleach, phenol (90%) and iodine disinfectants